Microarray fabrication system and method

ABSTRACT

A microarray is designed capture one or more molecules of interest at each of a plurality of sites on a substrate. The sites comprise base pads, such as polymer base pads, that promote the attachment of the molecules at the sites. The microarray may be made by one or more patterning techniques to create a layout of base pads in a desired pattern. Further, the microarrays may include features to encourage clonality at the sites.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/552,712, entitled “SINGLE MOLECULE MICROARRAY SYSTEM AND METHOD,”filed Oct. 28, 2011, which is herein incorporated in its entirety byreference for all purposes.

BACKGROUND

The invention relates generally to the field of microarrays used fordetecting and analyzing molecules of interest, particularly biologicalmaterials.

Biological microarrays have become a key mechanism in a wide range oftools used to detect and analyze molecules, including deoxyribonucleicacid (DNA) and ribonucleic acid (RNA). In these applications, themicroarrays are engineered to include probes for these nucleotidesequences present in genes in humans and other organisms. In certainapplications, for example, individual DNA and RNA probes may be attachedat small locations in a geometric grid (or randomly) on a microarraysupport. A test sample, such as from a known person or organism, may beexposed to the grid, such that complimentary genes of fragmentshybridize to probes at the individual sites in the array. The array canthen be examined by scanning specific frequencies of light over thesites to identify which genes or fragments in the sample are present, byfluorescence of the sites at which genes or fragments hybridized.

In similar applications, biological microarrays may be used for geneticsequencing and similar applications. In general, genetic sequencingconsists of determining the order of nucleotides or nucleic acid in alength of genetic material, such as a fragment of DNA or RNA.Increasingly longer sequences of base pairs are being analyzed, and theresulting sequence information may be used in various bioinformaticsmethods to logically fit fragments together so as to reliably determinethe sequence of much more extensive lengths of genetic material fromwhich the fragments were derived. Automated, computer-based examinationof characteristic fragments have been developed, and have been used morerecently in genome mapping, identification of genes and their function,evaluation of risks of certain conditions and disease states, and soforth. Beyond these applications, such microarrays may be used for thedetection and evaluation of a wide range of molecules, families ofmolecules, genetic expression levels, single nucleotide polymorphisms,and genotyping.

For these and other applications of biological microarrays, improvementshave recently been made in imaging systems for capturing data related tothe individual molecules attached at sites of the microarrays. Forexample, improvements in imaging systems allow for faster, more accurateand higher resolution scanning and imaging, particularly through the useof line-scanning and confocal control of imaging optics. However, as thedensity of microarrays increases, and the size of the areas containingindividually characterized sites also increases, scanning, both by pointscanning and line scanning approaches becomes problematic. Inparticular, there is a continuous drive in the field for more denselypacked arrays that can hold more molecular information on a givensupport (capable of being analyzed in a single text). This packingdensity poses challenges for both processing and imaging. Moreover, itwould be beneficial to provide a high degree of uniformity in themolecules attached at each site of the arrays, such that bettersignal-to-noise ratios are obtained for the individual sites. Currenttechniques for creating, preparing and utilizing the microarrays are inneed of improvement if further density and signal-to-noise improvementsare to be realized.

BRIEF DESCRIPTION

Embodiments of the present disclosure include a method for preparing abiological microarray. The steps of the method include forming an arrayof base pads at predetermined sites on a substrate, wherein individualbase pads are configured to capture a nucleic acid molecule; disposing anucleic acid molecule capture substance over each of the base pads; anddisposing a porous attachment layer over the base pads, wherein theporous attachment layer is configured to attach amplified copies of thenucleic acid molecules.

Embodiments of the present disclosure include a method for preparing abiological microarray. The steps of the method include providing anarray of base pads at predetermined sites on a substrate, whereinindividual base pads are configured to capture a nucleic acid molecule;and contacting the array of base pads with a mixture of differentnucleic acid molecules under conditions wherein a nucleic acid moleculeis captured at each base pad, wherein a porous attachment layer isdisposed over the base pads and the porous attachment layer isconfigured to attach amplified copies of the nucleic acid moleculescomprising nucleotides or nucleotide-like components.

Embodiments of the present disclosure include a method for preparing abiological microarray. The steps of the method include forming an arrayof base pads at predetermined sites on a substrate; disposing a moleculebinding substance over each of the base pads, thereby configuring eachof the base pads to capture a nucleic acid molecule; disposing a porousattachment layer over the base pads; seeding each of the base pads witha single nucleic acid molecule by linking the single nucleic acidmolecule to the molecule binding substance; and amplifying the nucleicacid molecule at each base pad to obtain at each base pad a regioncomprising copies of the nucleic acid molecule, wherein the copies ofthe nucleic acid molecule are attached to the porous attachment layer.

Embodiments of the present disclosure include a biological microarraysystem that includes an array of base pads at predetermined sites on asubstrate; a molecule binding substance disposed over each of the basepads configured to capture a nucleic acid molecule at each of the basepads; and a porous attachment layer disposed over the base pads, whereinthe porous attachment layer is configured to attach amplified copies ofthe nucleic acid molecules.

Embodiments of the present disclosure include a biological microarraysystem that includes an array of base pads at predetermined sites on asubstrate; a molecule binding substance disposed over each of the basepads and linked to no more than a nucleic acid molecule; a porousattachment layer disposed over the base pads; and several copies of eachof the nucleic acid molecules linked to the porous attachment layerdisposed over each of the respective base pads.

Embodiments of the present disclosure include a method for preparing abiological microarray. The steps of the method include forming a polymerlayer on a substrate; disposing a photoresist layer over the polymerlayer; forming interstitial spaces in the photoresist layer and thepolymer layer; removing the photoresist layer to expose polymer basepads, wherein the polymer base pads are coupled to a molecule bindingsubstance. The polymer layer may include apoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) polymer.

Embodiments of the present disclosure include a method for preparing abiological microarray. The steps of the method include activatingregions on a substrate to form a pattern of activated regions;contacting the substrate with a self-assembling monomer solution;polymerizing the monomers to form polymer base pads only on theactivated regions wherein the polymer base pads are coupled to amolecule binding substance.

Embodiments of the present disclosure include a method for preparing abiological microarray. The steps of the method include coupling aminegroups to a surface of a microarray substrate with a silylation reagent;coupling the amine groups to N-hydroxysulfosuccinimidyl-4-azidobenzoate;exposing the N-hydroxysulfosuccinimidyl-4-azidobenzoate to light suchthat a nitrene is generated; reacting the nitrene withpoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) monomers; andcross-linking thepoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) monomers toform a polymer. As an alternative to silylation reagent, polylysine orpolyethyleneimine can be used.

Embodiments of the present disclosure include a biological microarraysystem that includes an array of base pads at predetermined sites on asubstrate; a molecule binding substance disposed over each of the basepads; and a passivation layer disposed on the substrate between basepads. The passivation layer may include diamond-like carbon,hexa-methyldisilizane, Teflon, fluorocarbon, a polymer such aspolyethylene glycol (PEG) and/or Parylene.

Embodiments of the present disclosure include a method for preparing abiological microarray. The steps of the method include forming wells ona substrate, wherein the wells are separated by metal interstitialregions; applying a polymer layer on the substrate such that the polymerlayer covers the wells and the metal interstitial regions; cross-linkingthe polymer through the substrate; and removing the metal to yield asubstrate and a plurality of polymer pads coupled to a surface of thesubstrate, wherein the polymer pads comprise a molecule bindingsubstance. The metal interstitial regions can be configured as pillarsin some embodiments. Alternatively or additionally the interstitialregions can form a flat surface into which the wells form depressions.

Embodiments of the present disclosure include a method for preparing abiological microarray. The steps of the method include forming anelectrically conductive layer on a surface of a substrate; forming aplurality of spaced apart electrically nonconductive regions on theelectrically conductive layer; forming a polymer layer over theelectrically conductive layer and the plurality of spaced apartelectrically nonconductive regions, wherein the polymer is coupled to aplurality of primers; and applying a current through the electricallyconductive layer to deactivate only a portion of the primers coupled tothe polymer.

Embodiments of the present disclosure include a method for preparing abiological microarray. The steps of the method include forming a polymerlayer on a surface of a substrate; forming a plurality of spaced apartphotoresist regions on the polymer; contacting exposed portions of thepolymer layer with a plurality of primers; and removing the photoresistregions and covered portions of the polymer layer such that a pluralityof spaced apart polymer pads coupled the plurality of primers remain onthe surface of the substrate.

Embodiments of the present disclosure include a method for preparing abiological microarray. The steps of the method include forming wells ona substrate, wherein the wells are separated by photoresist interstitialregions; applying nanoparticles on the substrate such that thenanoparticles cover the wells and the photoresist interstitial regions;removing the photoresist such that a plurality of spaced apartnanoparticles remain on the surface of the substrate; and coupling amolecule binding substance to the nanoparticles. The photoresistinterstitial regions can be configured as pillars in some embodiments.Alternatively or additionally the interstitial regions can form a flatsurface into which the wells form depressions.

Embodiments of the present disclosure include a method for preparing abiological microarray. The steps of the method include (a) providing anamplification reagent comprising (i) an array of amplification sites,and (ii) a solution comprising a plurality of different target nucleicacids, wherein the different target nucleic acids have fluidic access tothe plurality of amplification sites and wherein the solution comprisesa molecular crowding agent such as a solution of at least 3% PEG. Themethod also includes reacting the amplification reagent to produce aplurality of amplification sites that each comprise a clonal populationof amplicons from an individual target nucleic acid from the solution,wherein the reacting comprises (i) producing a first amplicon from anindividual target nucleic acid that transports to each of theamplification sites, and (ii) producing subsequent amplicons from theindividual target nucleic acid that transports to each of theamplification sites or from the first amplicon.

Embodiments of the present disclosure include a method for preparing abiological microarray. The steps of the method include (a) providing anamplification reagent in a flow cell comprising (i) an array ofamplification sites, and (ii) a solution comprising a plurality ofdifferent target nucleic acids, wherein the different target nucleicacids have fluidic access to the plurality of amplification sites, and(b) applying an electric field across the flow cell to crowd the targetnucleic acids towards the array of amplification sites (c) reacting theamplification reagent to produce a plurality of amplification sites thateach comprise a clonal population of amplicons from an individual targetnucleic acid from the solution, wherein the reacting comprises (i)producing a first amplicon from an individual target nucleic acid thattransports to each of the amplification sites, and (ii) producingsubsequent amplicons from the individual target nucleic acid thattransports to each of the amplification sites or from the firstamplicon.

Embodiments of the present disclosure include a biological microarraysystem that includes an array of base pads at predetermined sites on asubstrate; a molecule binding substance disposed over each of the basepads; and a dendron coupled to each of the base pads, wherein thedendron comprises a plurality of ends and wherein the plurality of endsare functionalized with binding groups.

Embodiments of the present disclosure include a biological microarraysystem that includes an array of base pads at predetermined sites on asubstrate; and at least one primer coupled to each of the base pads,wherein a first portion of the primers are coupled to the base pad at afirst end and wherein a second portion of the primers are coupled to thebase pads at a second end, wherein the first end comprises a cleavableportion.

Embodiments of the present disclosure include a biological microarraysystem that includes an array of base pads at predetermined sites on asubstrate; a layer of silane-free acrylamide disposed on the substratebetween the array of base pads, wherein the layer of silane-freeacrylamide comprises a plurality of primers comprises a first adapterend and a second adapter end; and a second plurality of primers coupledto the base pads such that at least one primer is coupled to each of thebase pads, wherein second plurality of primers comprises the firstadapter end and a third adapter end.

Embodiments of the present techniques are described herein by referenceto a microarray for use with a biological analysis device. Thedisclosure is not, however, limited by the advantages of theaforementioned embodiment. The present techniques may also be applied todevices capable of generating other types of biological data or forother types of molecule capture. Further, it should be understood thatthe disclosed embodiments may be combined with one another. In addition,features of particular embodiments may be exchanged with features ofother embodiments.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1 is a diagrammatical representation of an exemplary microarrayaccording to the present disclosure, illustrating the overall layout ofthe microarray and detailing the arrangement of individual sites;

FIG. 2 is a diagrammatical representation of general phases in themanufacturing, preparation and use of such microarrays;

FIGS. 3-8 are diagrammatical representations of successive steps in thedisposition of sites on a substrate for one of the microarrays;

FIGS. 9-11 are diagrammatical representations of steps in thepreparation of sites of the exemplary microarray once formed;

FIGS. 12 and 13 are diagrammatical representations of capture andamplification techniques for use with the exemplary microarray;

FIG. 14 is a diagrammatical representation of steps in the preparationof base pads in accordance with embodiments of the present techniques;

FIG. 15 is a diagrammatical representation of reactive pads formed in asubstrate in accordance with embodiments of the present techniques;

FIG. 16 is a diagrammatical representation of steps in the preparationof a polymer layer in accordance with embodiments of the presenttechniques;

FIG. 17 is a diagrammatical representation of steps in the preparationof a polymer layer in accordance with embodiments of the presenttechniques;

FIG. 18 is a diagrammatical representation of steps in the preparationof a polymer layer in accordance with embodiments of the presenttechniques;

FIG. 19 is a diagrammatical representation of steps in the preparationof a polymer layer in accordance with embodiments of the presenttechniques;

FIG. 20 is a diagrammatical representation of steps in the preparationof a polymer layer in accordance with embodiments of the presenttechniques;

FIG. 21 is a diagrammatical representation of steps in the preparationof base pads in accordance with embodiments of the present techniques;

FIG. 22 is a diagrammatical representation of steps in the preparationof base pads in accordance with embodiments of the present techniques;

FIG. 23 is a diagrammatical representation of steps in the preparationof base pads in accordance with embodiments of the present techniques;

FIG. 24 is a diagrammatical representation of steps in the preparationof base pads in accordance with embodiments of the present techniques;

FIG. 25 is a diagrammatical representation of steps in the preparationof base pads and wells in accordance with embodiments of the presenttechniques;

FIG. 26 is a diagrammatical representation of steps in the preparationof primer patterns in accordance with embodiments of the presenttechniques;

FIG. 27 is a diagrammatical representation of steps in the preparationof base pads in accordance with embodiments of the present techniques;

FIG. 28 is a diagrammatical representation of steps in the preparationof base pads in accordance with embodiments of the present techniques;

FIG. 29 is a diagrammatical representation of steps in the preparationof nanoparticles in accordance with embodiments of the presenttechniques;

FIG. 30 is a diagrammatical representation of steps in the preparationof nanoparticles in accordance with embodiments of the presenttechniques;

FIG. 31 is a diagrammatical representation of steps in the deposition ofmicelles in accordance with embodiments of the present techniques;

FIGS. 32A and 32B are cluster image data of a diamond-like carbonpatterned microarray;

FIG. 33 is a diagrammatical representation of steps in the preparationof a diamond-like carbon passivation layer in accordance withembodiments of the present techniques;

FIG. 34A and FIG. 34B illustrate PEG molecular crowding results with andwithout PEG;

FIG. 35 is a diagrammatical representation of steps involved inconcentrating DNA via an electrical field in accordance with embodimentsof the present techniques;

FIG. 36 is an exemplary circuit that may be used with an electricallyconductive flow cell

FIG. 37 is an exploded view of a flow cell including an electricallyconductive layer in accordance with embodiments of the presenttechniques;

FIG. 38 is a perspective view of a flow cell configured fordielectrophoresis in accordance with embodiments of the presenttechniques;

FIG. 39 is a schematic diagram of electric fields generated by the flowcell of FIG. 39;

FIG. 40 is a diagrammatical representation of steps involved ingenerating primers with dendron termini in accordance with embodimentsof the present techniques;

FIG. 41 is a diagrammatical representation of steps involved in formingclusters with primers captured at both ends in accordance withembodiments of the present techniques;

FIG. 42 is a diagrammatical representation of steps involved in formingsites with characteristic end primers and interstitial spaces withprimers with different characteristic end primers;

FIG. 43 is a diagrammatical representation of cluster formation in thesites of FIG. 43; and

FIG. 44 is a flow chart illustrating exemplary steps in the use with anexemplary microarray.

DETAILED DESCRIPTION

The present disclosure provides improved techniques for making andutilizing microarrays. The techniques may draw upon a range of differenttechnologies for creating a prepared microarray ready to receivemolecules of interest for analysis. The microarrays offered areparticularly suited for capturing one or more molecules of interest ateach site, and these molecules may be subsequently amplified to providea generally uniform probe of the same molecule at the individual sites.The techniques may be used for microarray analysis and/or sequencing,such as sequencing of DNA and RNA (including cDNA. In certainembodiments, the techniques may be used with a variety of sequencingapproaches or technologies, including techniques often referred to assequencing-by-synthesis (SBS), sequencing-by-ligation, pyrosequencingand so forth.

Turning now to the drawings, and referring first to FIG. 1, an exemplarymicroarray 10 is illustrated for detecting and analyzing molecules ofinterest. In general, the microarray comprises a substrate 12 andsections 14 separated by open areas. The sections 14 may each compriseregions 16, which may generally form lines across the substrate. Each ofthese regions, in turn, comprises multiple domains 18, each separatedfrom one another by open areas 20. Finally, the domains 18 comprisemultiple individual sites 22 where the molecules of interest will bedeposited and attached for analysis. As noted below, many differentlayouts of the sites may be envisaged, including regular, repeating, andnon-regular patterns. In a presently contemplated embodiment, forexample, the sites are disposed in a hexagonal grid for close packingand improved density. Other layouts may include, for example,rectilinear (i.e., rectangular) layouts, triangular layouts, and soforth. The particular layouts, and differences between the layouts ofdifferent domains, if used, may follow the teachings of U.S. Pat. No.7,813,013, and/or of U.S. patent application Ser. No. 13/267,565, filedon Oct. 6, 2011 which are hereby incorporated by reference in itsentirety. It should be noted that the patterned substrate (microarray)may also be used to control the density of the features capable ofinterrogation (e.g., through imaging). In addition to enabling acontrolled increase in density, the present techniques also provide ameans to control a lower density regime. It should also be noted that,as discussed below, the microarray illustrated and discussed in thepresent disclosure will typically be disposed in or formed as a part ofa flow cell in which various carrier fluids, reagents, and so forth maybe introduced. Moreover, the particular orientation of the features,sites, sections, domains and so forth may differ from those illustratedin FIG. 1. In some embodiments, the sections 14, regions 16, domains 18and/or sites 22 are contiguous and thus need not be separated by openareas.

In many cases, the microarray will be used to analyze biologicalmolecules, such as nucleotides, oligonucleotides, nucleic acids, aminoacids, polypeptides, proteins, and other bioactive reagents at thesites, that may be prepared in advance. The resulting system may bedesigned for synthesizing one or more of the above biopolymers orsequencing such biopolymers. It should be borne in mind that the presenttechniques although useful for sequencing operations, gene expressionoperations, diagnostic applications, diagnostic applications, or any oneof these, are not necessarily limited to those uses. For example themethods and compositions set forth herein may be used for manufacturing,preparing, imaging, and analyzing collected image data for any desiredapplication

The disclosed embodiments may be used with any known combinatorialchemistry or biochemistry assay process, and are especially adaptable toassays having solid phase immobilization. For example, the disclosedembodiments may be used in many areas such as drug discovery,functionalized substrates, biology, proteomics, combinatorial chemistry,and any assays or multiplexed experiments. Examples of common assays areSNP (single nucleotide polymorphism) detection, DNA/genomic sequenceanalysis, genotyping, gene expression assays, proteomics assay, peptideassays, antigen/antibody assays (immunoassay), ligand/receptor assays,DNA analysis/tracking/sorting/tagging, as well as tagging of molecules,biological particles, cell identification and sorting, matrix supportmaterials, receptor binding assays, scintillation proximity assays,radioactive or non-radioactive proximity assays, and other assays, highthroughput drug/genome screening, and/or massively parallel assayapplications. The analyte of interest may be labeled, detected oridentified with any technique capable of being used in an assay witharrays or beads, including but not limited to fluorescent, luminescent,phosphorescent, quantum dot, light scattering colloidal particles,radioactive isotopes, mass spectroscopy, NMR (nuclear magneticresonance), EPR (electro paramagnetic resonance), ESR (electron spinresonance), IR (infrared), FTIR (Fourier transform infra red), Ramanspectroscopy, or other magnetic, vibrational, electromagnetic,electrical, pH, chemical or optical labeling or detection techniques.Optical or non-optical detection techniques and optionally optical ornon-optical labels can be used in a method or composition set forthherein. The invention provides array surfaces having the disclosedcoatings and/or features.

In the illustrated embodiment, however, exemplary biological moleculesmight include, but are not limited to, any of a variety of moleculesthat have a biological activity or are reactive with biological systems.Examples include nucleic acids, such as DNA, RNA or analogs of DNA orRNA. Other exemplary biological molecules might include proteins (alsoreferred to as polypeptides), polysaccharides or analogs thereof.Exemplary proteins include, but are not limited to, nucleicacid-specific proteins such as polymerases, transcription factors,single stranded binding proteins or restriction endonucleases; lectins;or avidin or analogs thereof. Other biological molecules include SNAREpeptides, aptamers and ribosomes. The methods and compositions set forthherein need not be limited to analyzing biological molecules, beinguseful for example, with other types of biological materials such ascells or sub cellular particles such as organelles. Molecules andmaterials other than biological molecules and materials may be analyzedas well.

Although any of a variety of biopolymers may be used, for the sake ofclarity, the systems and methods used for processing and imaging in theexemplary context illustrated in FIG. 1 and elsewhere herein will bedescribed with regard to processing of nucleic acids. In general, themicroarray of FIG. 1 comprises probes that may include one reaction siteor an array of reaction sites. As used herein, the term “array” or“microarray” refers to a population of individual reaction sites on oneor more substrates such that individual reaction sites may bedifferentiated from each other according to their relative location.Ideally, a single species of biopolymer may be attached to eachindividual reaction site, and the techniques described below facilitatesuch individualization. Moreover, multiple copies of particular speciesof biopolymer may be attached to a particular reaction site, such as byamplification of a single molecule or multiple molecules initiallycaptured or seeded at the site. The array taken as a whole willtypically include a plurality of different biopolymers, e.g., aplurality of clonal copies attached at a plurality of different sites.The reaction sites may be located at different addressable locations onthe same substrate, and in many applications, such addressing, andindexing of the particular sites for subsequent data analysis, arecarried on during the processing of the prepared microarray (e.g.,imaging and image analysis).

In general, the microarrays made and used as set forth in the presentdisclosure will be intended, in many applications, for analyzing nucleicacids. As will be appreciated by those skilled in the art, suchmolecules will often be of interest in certain naturally occurringcontexts, such as chromosomal and non-chromosomal DNA of living beings(humans, animals, plants, microbes, and so forth). However, as usedherein, the term “nucleic acid” should be considered to include bothnaturally and non-naturally occurring variants.

Further, certain embodiments of the present disclosure relate to captureof a single molecule of interest per site on a microarray. This may beachieved by any suitable technique, such as via size exclusion. Inaddition, certain embodiments of the present disclosure may relate tothe capture of multiple molecules of interest. For example, kineticexclusion techniques may permit capture of multiple molecules ofinterest. Kinetic exclusion can exploit conditions that yield arelatively slow rate of target nucleic acid capture vs. a relativelyrapid rate for making copies of the target nucleic acid. Alternativelyor additionally, kinetic exclusion can exploit a relatively slow ratefor making a first copy of a target nucleic acid vs. a relatively rapidrate for making subsequent copies of the target nucleic acid or of thefirst copy. In one embodiment, although an individual site may have beenseeded with several different target nucleic acids, kinetic exclusionwill allow only one of those target nucleic acids to be amplified. Morespecifically, once a first target nucleic acid has been activated foramplification, the site will rapidly fill to capacity with its copies,thereby preventing copies of a second target nucleic acid from beingmade at the site. Kinetic exclusion techniques such as those disclosedin U.S. Provisional Application No. 61/660,487, which is incorporated byreference in its entirety herein for all purposes, may be used inconjunction with the disclosed embodiments.

FIG. 2 generally represents certain phases included in the manufacture,preparation, and use of a microarray in accordance with the presentdisclosure. The microarrays may be formed from a blank 24 during asubstrate preparation phase 26. The blank may be made of any suitablematerial, such as glass. Other suitable substrate materials may includepolymeric materials, plastics, silicon, quartz (fused silica), borofloatglass, sapphire, plastic materials such as COCs and epoxies. The surfacepreparation phase 26 may include processes that predispose the blank 24for efficient downstream processes such as site formation, sitepreparation, and molecule capture and preparation. The blank 24 is cutor sliced into substrate dies 28 which may generally have the form ofthe microarray. This initial substrate preparation phase is thenfollowed by a site formation phase 30 in which the individual sites 32are formed on the substrate. It should be noted that the operations maybe performed in different orders and manners. For example, in apresently contemplated method, the array of capture sites are applied toa blank substrate prior to cutting the substrate from a wafer or blankthat is used to form many microarrays. Functionalization of the capturesites, as described below, is performed after the cutting operation inthis particular embodiment. A range of different techniques arepresently contemplated for formation of the sites. One of thesetechniques is adapted to dispose a material at each site location thatmay be built upon for accommodating the molecule capture andamplification desired. Exemplary techniques include nano-imprintlithography, described in greater detail below, as well as dip penlithography, photolithography, and micelle lithography. In one presentlycontemplated embodiment, the sites are formed by deposition of a basepad at each site location. The site pads may be made of any suitablematerial, such as gold or another metal. Other suitable material mayinclude silanes, functional biomolecules such as avidin orfunctionalized organic or inorganic molecules, titanium, nickel, andcopper. Alternatively, the site pads may be created by simply blockingthe interstitial space with a resist or chemical moiety that resistsattachment of a binding moiety leaving the site pad composed of nativesubstrate material (i.e. glass, etc). The site pads can then bederivatized with binding moieties that react specifically with thesubstrate material (i.e. glass, etc.) and not interstitial space. Itshould be noted that the array of base pads could be an array ofnanodots or nanoparticles. Further, the substrate may include any numberand/or arrangement of image registration features.

Once the sites are laid out on the substrate, site preparation mayproceed as indicated at reference numeral 34, resulting in a preparedmicroarray 36 ready to be further processed to receive a sample ofmolecules to be tested. This phase of the manufacturing process mayinclude deposition of various materials on the pads, but also around thepads or over the entire extent of the substrate. These materials may beadapted to enhance the capture of one or more molecules at each sitelocation, and optionally for subsequently amplifying the molecules forfurther reading analysis. In the exemplary embodiment, substratepreparation phase 26, the site formation phase 30, and the sitepreparation phase 34 may be thought of as the major steps in themanufacturing of the microarray. Thereafter, the microarray may bestored and utilized as described below. Moreover, any of theintermediate preparation stages may be performed by the same or separateentities, with intermediate products being further processed to arriveat the final prepared microarray. It should also be noted that whilemicroarrays having a single prepared surface are illustrated anddescribed here, as discussed below, the microarrays may be used inapplications where more than one surface is prepared and used formolecule captures, amplification, reading and analysis. Moreover, themicroarrays may typically be disposed in a flow cell that permits theintroduction of chemistry useful for adding nucleotides and othersubstances, templates for reading, sequencing, and so forth, agents fordeblocking locations on the templates, washing and flushing liquids, andso forth. Such flow cells are described, for example, in U.S. patentapplication publication no. US 2010/0111768 A1 and U.S. Ser. No.13/273,666, each of which is hereby incorporated by reference in itsentirety.

Once prepared for use, the microarray may be employed to capture one ormore molecules at each site location as indicated by phase 38 in FIG. 2.The molecule or molecules will typically be amplified, such as by bridgeamplification, although other amplification processes may also be used.For example, amplification of a template nucleic acid may be carried outusing bridge amplification as described in Bentley et al., Nature456:53-59 (2008); U.S. Pat. No. 5,641,658 or 7,115,400; or in U.S. Pat.Pub. Nos. 2002/0055100 A1, 2004/0096853 A1, 2004/0002090 A1,2007/0128624 A1, or 2008/0009420 A1, each of which is incorporatedherein by reference in its entirety. In this example, the bridgeamplification may be primed by primer nucleic acids that are attached toa porous attachment layer that is in contact with a base pad to which atemplate nucleic acid is attached. Thus, the base pad can seed growth ofa cluster of nucleic acid copies of the template that forms in theporous attachment layer around the base pad.

Another useful method for amplifying nucleic acids is rolling circleamplification (RCA). RCA may be carried out, for example, as describedin Lizardi et al., Nat. Genet. 19:225-232 (1998) or US Pat. Pub. No.2007/0099208 A1, each of which is incorporated herein by reference inits entirety. Also useful is multiple displacement amplification (MDA),for example, using a product of RCA (i.e. an RCA amplicon) as atemplate. Exemplary methods of MDA are described in U.S. Pat. Nos.6,124,120; 5,871,921; or EP 0,868,530 B1, each of which is incorporatedherein by reference in its entirety. In embodiments that include anamplification step, one or more primers that are used for amplificationmay be attached to a base pad or the porous attachment layer. Theprimers need not be attached to a base pad or a porous attachment layerin some embodiments.

A molecule that is captured at a site or otherwise used in a method orcomposition herein may be a nucleic acid that is single stranded ordouble stranded. Typically the nucleic acid will have a single copy of atarget sequence of interest. Nucleic acids having concatameric copies ofa particular sequence may be used (e.g. products of rolling circleamplification). However, in many embodiments the nucleic acid will nothave concatameric copies of a sequence that is at least 100 nucleotideslong or that is otherwise considered a target sequence for a particularapplication of the methods. Although the methods and compositions areexemplified with respect to capture of a nucleic acid molecule, it willbe understood that other molecules and materials such as those set forthabove in regard to microarray analysis can also be captured at a site orotherwise used.

The prepared microarray with the probes attached, as indicated byreference numeral 40, may then be used for analysis purposes. Thereading/processing phase 42 is intended to include the imaging of themicroarray, the use of the image data for analysis of the moleculescaptured and amplified at each of the sites, and so forth. More will besaid about this reading/processing phase below. The entire processingsystem denoted generally by reference numeral 44 in FIG. 2, may includevarious imagers, readers, data analysis systems, and so forth asdescribed generally in U.S. Pat. No. 7,329,860; U.S. patent applicationpublication nos. US 2010/0111768 A1, or 2011/0220775 A1; or U.S. Ser.No. 61/438,486 or 13/006,206, each of which is hereby incorporated byreference in its entirety.

As mentioned above, one presently contemplated approach for forming thebase pads or site locations on substrate involves large-area patterningof very small features using techniques such as nanoscale imprintlithography. FIGS. 3-8 illustrate exemplary steps in an imprintlithography process. Referring first to FIG. 3, a substrate die 28 isfirst coated with a transfer layer 46, such as by spin or spray coating.This layer may be formed of a commercially available resist, such aschlorobenzene and a methylacrylate polymer, and may have a nominalthickness of approximately 70 nm. On this transfer layer, an ultraviolet(UV) imprint resist layer 48 is disposed. This layer also may be formedby a polymer which may be spin or spray coated on the transfer layer.This UV imprint resisted layer will form an etch barrier in subsequentprocessing. This layer may be formed, for example, of tert-butylmethylacrylate and polyester modified polydimethylsiloxane and polyesteracrylate and a photo-initiator, at a nominal thickness of approximately10 nm thicker than the feature height on the working mold 50, typically70 nm. A working mold 50 is formed in advance, and may be made ofvarious materials, such as glass or modified polydemethylsiloxane. Theworking mold will be generally transparent to UV light, to permit curingas described below. The desired pattern for the site pad will be formedin the working mold, such that recesses 52 will separate lands 54. Therecesses 52 will generally correspond to spaces that will be formedaround the pads on the substrate, while the lands 54 in this embodimentwill generally correspond to the locations of the pads. The size of theseparated lands may be tuned and can range, for example, from 5 nm(nanometers) to 3 μm (micrometers).

As illustrated in FIG. 4, during processing the mold is brought intocontact with the UV imprint layer and displaces portions of this layerto form regions 56 within the recesses 52 of the mold. That is, thelands 54 displace the UV imprint resist layer such that the lands aregenerally adjacent to the underlying transfer layer. With the mold inplace, then, the structure is exposed to UV radiation to at leastpartially cure the regions 56, rendering them resistant to subsequentetching and effectively transferring the pattern on the working moldinto the resist. With the mold then removed, as illustrated in FIG. 5,the transfer layer 46 remains on the substrate die 28, and the remainingregions 56 of the UV imprint resist layer remain to protect theunderlying regions of the transfer layer. Exposed transfer regions 60remain at what will become the locations of the site pads. An etchprocess is then used to remove these regions as illustrated in FIG. 6.Once the exposed transfer regions are removed, exposed substrate regions62 will remain. Subsequently, the structure is subjected to a depositionprocess, such as a metal deposition, to deposit a layer of material 64over both the regions 56 and the exposed substrate regions 62. In acurrently contemplated embodiment, the deposition is of a thin layer ofgold, although other materials may include Al, Al₂O₃, Zn, ZnO, Ni, Ti,TiO₂, ITO (Indium tin oxide), etc. Moreover, the deposition may be toany desired thickness, such as a nominal thickness of 5 nm. Finally, ina lift-off step, the layers above and below the regions 56, includingthese regions themselves are removed to leave only the pads at locations32 and the substrate. This lift-off operation may involve solventwashing steps and sonification. Following these processes, a substratedie will be provided with the sites determined and formed in the desiredpattern of sites, domains, regions, and so forth.

Once the sites are laid out and formed by positioning the site pads onthe substrate, subsequent building of the sites and preparation stepsmay take place. As illustrated in FIG. 9, in a presently contemplatedembodiment, each base pad 68 receives a capture substance 70 designed topromote the capture of a molecule of interest. FIG. 9, as with otherfigures in this disclosure, is not necessarily drawn to scale. Forexample, the capture substance may be submicroscopic in size (e.g. alinker molecule) or may be a particle that is, at least in some cases,visible under a microscope. In a presently contemplated embodiment, thesubstance comprises thiol-avidin, although other substances may beutilized, such as silanes, biotin-binding proteins, functionalbiomolecules such as avidin, streptavidin, neutravidin, andfunctionalized organic or inorganic molecules. An example is agold-patterned array functionalized with thiol-avidin to bind moleculesmodified with biotin. Other capture substances may include, for example,biological binding molecules including neutravidin, streptavidin,antibodies, etc., chemical binding moieties such as amines, aldehydes,carboxyl groups, etc.; and inorganic binding moieties such as metalchelates (i.e. histidine binding), gold (thiol binding), etc.

A capture substance may be attached to a base pad or site via a covalentor non-covalent linkage. Exemplary covalent linkages include, forexample, those that result from the use of click chemistry techniques.Exemplary non-covalent linkages include, but are not limited to,non-specific interactions (e.g. hydrogen bonding, ionic bonding, van derWaals interactions etc.) or specific interactions (e.g. affinityinteractions, receptor-ligand interactions, antibody-epitopeinteractions, avidin-biotin interactions, streptavidin-biotininteractions, lectin-carbohydrate interactions, etc.). Exemplarylinkages are set forth in U.S. Pat. Nos. 6,737,236; 7,259,258; 7,375,234and 7,427,678; and US Pat. Pub. No. 2011/0059865 A1, each of which isincorporated herein by reference.

As illustrated in FIG. 10, then, in a presently contemplated embodimenta charged layer 72 may be disposed over the pads and capture substance.In this embodiment, if used, the charged layer comprisesaminopropyltriethoxysilane (APTES). This charged layer may promote theattachment of the molecules at each site, while preventing attachmentwhere not desired. As illustrated in FIG. 11, an attachment layer 74 isdisposed over at least the pads 68, and in the illustrated embodimentmay be disposed over the entire substrate. In other embodiments, theattachment layer may be patterned such that it is present over the padsor sites but substantially absent over interstitial regions between thepads or sites.

An attachment layer used in a method or composition herein may be formedof a micro-porous material, such as silane-free acrylamide (SFA).Silane-free acrylamide (SFA) polymer may be formed by polymerization ofsilane free acrylamide and N—(S bromoacetamidylpentyl) acrylamide(BRAPA). Other attachment layers that may be used include withoutlimitation, acrylamide, methacrylamide, hydroxyethyl methacrylate,N-vinyl pyrrolidinone or derivatives thereof. Such materials are usefulfor preparing hydrogels. In some embodiments, the polymerizable materialcan include two or more different species of compound that form aco-polymer. Exemplary hydrogels and polymerizable materials that may beused to form hydrogels are described, for example, in US Pat. Pub. No.2011/0059865 A1, which is incorporated herein by reference in itsentirety. Other hydrogels include but are not limited to, polyacrylamidepolymers formed from acrylamide and an acrylic acid or an acrylic acidcontaining a vinyl group as described, for example, in WO 00/31148(incorporated herein by reference in its entirety); polyacrylamidepolymers formed from monomers that form [2+2] photo-cycloadditionreactions, for example, as described in WO 01/01143 or WO 03/014392(each of which is incorporated herein by reference in its entirety); orpolyacrylamide copolymers described in U.S. Pat. No. 6,465,178, WO01/62982 or WO 00/53812 (each of which is incorporated herein byreference in its entirety). PAZAM is also useful as set forth in furtherdetail below. The attachment layer can function to attach the moleculesand/or it can provide locations for attachment of identical molecules(i.e. copies of the molecules) at each site during amplification.

As noted above, various layouts may be envisaged for the sites of themicroarray. Moreover, the density, location, pitch, and sizes of thesites may vary depending upon such factors as the array design, the typeof processing and imaging equipment used for analyzing the arrays, andthe molecules to be processed. By way of example, presently contemplatedsites made as set forth in the present disclosure may have sizesdictated by the desired imaging and/or reaction modality. For example,sites may be approximately 30-500 nm and may be in a range of 30-300 nmor 300-500 nm. The sites may be disposed on the substrate in a hexagonalpattern. The sites may be present at a density of approximately 1million capture sites per square millimeter, but can easily be tuned byadjusting the pitch to densities greater than 5 million capture sitesper square millimeter. While the particular pitch of the sites may vary,depending, for example, upon their size and the density desired, typicalpitches may include at most about 5 micron, 2 micron 1 micron, 850 nm,or 750 nm, or even lower value.

The sites or pads used in various embodiments may be in a size rangethat is useful for capture of a single nucleic acid template molecule toseed subsequent formation of a homogenous colony, for example, viabridge amplification. FIG. 12 illustrates a base pad 68 that is attachedto a capture substance 70 that is in turn attached to a single nucleicacid template 76. The nucleic acid template is illustrated as extendingout of the attachment layer 74. However, in some embodiments the nucleicacid template may be retained under or within the volume of theattachment layer. Bridge amplification may be primed by primer nucleicacids that are attached to the attachment layer (e.g. the attachmentlayer may be a gel) to seed growth of a cluster of nucleic acid copiesof the template that forms in or on the attachment layer around the basepad 68.

In an exemplary bridge amplification method, a template nucleic acidhybridizes to a gel-attached primer and the 3′ end of the primer isextended to create a complementary copy of the template. In someembodiments two different primers may be attached to the gel. Theprimers can form a pair used for amplification of a template and itscomplementary copy. As such, two primers may be used for amplificationof the template into multiple copies to form a nucleic acid cluster orpopulation of amplicons. For example, amplification may be carried outusing bridge amplification to form nucleic acid clusters attached to thegel. Useful bridge amplification methods are described, for example, inU.S. Pat. Nos. 5,641,658 and 7,115,400; U.S. Pat. Pub. Nos. 2002/0055100A1, 2004/0096853 A1, 2004/0002090 A1, 2007/0128624 A1, and 2008/0009420A1, each of which is incorporated herein by reference in its entirety.Any of a variety of solid phase amplification techniques can be usedsuch as solid phase PCR (whether isothermal or thermocyclic) using afirst primer species that is solid phase attached and a second primerspecies that is in solution. Other useful methods for amplifying nucleicacids using one or more gel-attached primers are rolling circleamplification (RCA) and multiple displacement amplification (MDA).

In particular embodiments, a cluster of nucleic acids may have a footprint that is no larger than the area of the base pad. For example, theattachment layer 74 may be confined to the foot print of the base pad68. As such the base pad (and optionally the attachment layer) can forma cluster restriction zone along the lines illustrated in FIG. 13.Alternatively, the foot print of a cluster may be larger than the basepad 68 from which it was seeded.

One aspect of the present techniques disclosed herein relates to aprocess for preparing a polymer coating immobilized to a surface of asubstrate. In some embodiments, the method comprises polymerizing apolymerizable material, which may be any suitable polymer in accordancewith the present techniques, on a surface 90 of a substrate (e.g.,substrate die 28), wherein the surface comprises a plurality offunctional groups, thereby forming a layer of polymer coating over allor a part of the surface. The polymer coating can be covalently bondedto the functional or reactive groups on the surface. In certainembodiments, the microarrays may also use base pads 68 formed viaselective patterning as illustrated in FIG. 14, which represents stagesincluded in one example of the manufacture and preparation of amicroarray including base pads 68 in accordance with the presentdisclosure. Further, the disclosed techniques for surface patterning maybe used with other suitable site materials to form base pads, eitherwith or without polymers.

As illustrated in FIG. 14, the substrate die 28 is coated with a polymerlayer 100 (e.g., via spin coating or dunk coating) with one or morephotoresist layers 102 disposed over the polymer layer 100 such that thepolymer layer 100 is between the die 28 and the photoresist layer(s) 102at stage 104. After a photolithography step 106, the surface 90 of thesubstrate die 28 includes an intact polymer layer 100 and wells 108 inthe photoresist layer 102 after removal of a portion of the photoresistlayer 102 to expose portions 110 of the polymer layer 100 that will beremoved in subsequent steps. After an etching step 114 (e.g., reactiveion etching), portions 110 of the polymer layer 100 have been removed toexpose the surface 90 of the substrate die 28. After a liftoff step 116,the base pads 68 are in place on the surface 90 of the substrate die 28following liftoff of the remaining photoresist layer 102. Thepreparation of the base pads 68 may include one or more of lithography,imprint lithography, and etching steps. Further primer grafting can beperformed at the beginning, during or at the end of the proposedsequence, before photoresist deposition or can follow the exposure ofthe base pads 68 as a solution-based technique.

FIG. 15 is an example of an alternate technique for forming base pads68. In the depicted embodiment, the substrate die 28 is functionalizedto form chemically reactive pads 120 on the surface 90 having thedesired pattern. For example, if the substrate die 28 is glass, thereactive pads may be reactive silane pads. The polymer formation islimited to only the reactive portions of the substrate die 28. Thesubstrate die 28 may be formed first, and the reactive pads 120 may befunctionalized by any suitable patterning technique, such as thephotolithography, etching, and or masking techniques provided herein.

FIG. 16 is a schematic depiction of the formation of a polymer brushpolymer on a reactive pad 120. A self-assembling monomer layer 124 iscontacted with the reactive pad 120 at step 126. The monomers formcovalent bonds with the reactive pads 120 at step 128 and then arepolymerized at step 130 to form a polymer brush 132. The polymer padsare then directly grown from the chemically reactive pads 120. In thedepicted example, primer grafting may be done before, during or afterpolymerization is complete and may be a homogenous or heterogeneousreaction.

In one or more of the embodiments set forth herein, the polymer may be apoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM)polymer. For example, such polymers may be those disclosed in U.S.Provisional Application No. 61/657,508. In one specific embodiment, thepolymer comprises a polymer of Formula (I)

where n is an integer in the range of 1-10,000, and m is an integer inthe range of 1-10,000. Further, in one embodiment, the molecular weightof the polymer may be about 300 kDa to 500 kD, or, in a specificembodiment, about 312 kDa. In embodiments in which a PAZAM polymer isimplemented, polymerization may take place via a surface initiated atomtransfer radical polymerization (SI-ATRP) (as shown in FIG. 17) to asilanized surface. As shown in FIG. 17, the surface is pre-treated withAPTS (methoxy or ethyoxy silane) to covalently link silicon to one ormore oxygen atoms on the surface (without intending to be held bymechanism, each silicon may bond to one, two or three oxygen atoms asindicated by the generic bonding structure in FIG. 17). This chemicallytreated surface is baked to form an amine group monolayer. The aminegroups are then reacted with Sulfo-HSAB to form an azido derivative. UVactivation at 21 degrees C. with 1 to 30 J/cm² of energy generates theactive nitrene species, which can readily undergo a variety of insertionreactions with the PAZAM. In the depicted embodiment, the polymer mayinclude a Br precursor to the azide chain that acts as a cross-linker.

FIG. 18 is a reaction diagram of UV-mediated linking of PAZAM monomersto an amine-functionalized surface, such as those generated via SI-ATRPreactions. The reaction begins with linking a photoactive coupling agentN-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HSAB). Sulfo-HSAB is acommercially available bifunctional crosslinking agent including aphotoactive aryl azide and an activated NHS unit. Upon exposure to UVlight (250-374 nm), the aryl azide generates a nitrene with the releaseof nitrogen. This highly reactive species can undergo a variety of rapidinsertion reactions. As illustrated in FIG. 19, after the photoactiveunit is attached, the PAZAM is deposited (e.g., via open wafer orflowthrough), followed by UV irradiation and linking.

FIG. 20 is an alternative thermal linkage reaction for linking PAZAM tothe substrate die 28 (e.g., via chemically reactive pads 120). In thedepicted embodiment, the reaction begins by thermally linking the activegroup (acryloyl chloride or other alkene or alkyne-containing molecule)with subsequent deposition of PAZAM and application of heat. It iscontemplated that the thermal linkage reaction may yield a mixture ofthe 1,4 and 1,5 isomers and also adducts resulting from the 1,4 additionto the conjugated alkene moiety.

In addition to approaches in which a polymer layer is applied directlyto the substrate die surface by growing the polymer layer in place, amicrocontact printing approach is also contemplated. This approach,shown in FIG. 21, uses a soft or hard stamp 150 that has pillars 152coated with a patterning medium 154. The medium may include polymers(including PAZAM), reactants, binders, surfactants, and/or catalysts.The stamp selectively delivers the patterning medium to defined regionson the substrate die 28. As shown, the surface 90 of the substrate die28 may include an alkyne-APTES layer 158 or suitable reactive mediumonto which the base pads 68 (or other types of base pads) are applied.The result is a patterned substrate die 28 including base pads, such asbase pads 68 that support DNA cluster formation sequencing. In thedepicted embodiment, primers may be grafted before, during or afterpatterning, could be present in the patterning medium, and could begrafted via homogenous or heterogeneous reactions.

It is contemplated that the base pads 68 (including, but not limited to,PAZAM polymers) are coupled to the substrate die 28 via covalent ornon-covalent attachment protocols. In any of the disclosed embodiments,a photoresist material may protect the interstitial regions of thesubstrate die 28 from reacting/absorbing the polymer that is appliedduring formation of the base pads 68. A liftoff of the photoresistprotective layer leaves behind only surface-attached polymer. Primergrafting to the base pads 68 for subsequent molecule capture may followvia homogeneous or heterogeneous methods.

FIG. 22 illustrates various stages in one embodiment of a PAZAM base pad68 attachment technique using a sulfo-HSAB photoactive coupler. At stage160, the photoresist layer 162 is deposited on a reactive surface 164 ofthe substrate die 28. The photoresist layer 162, as depicted, formsreactive wells 170 and interstitial regions 172 that are elevatedrelative to the wells (e.g. forming pillars). After application of thecoupling agent 176 (stage 180), which covers the wells 170 and theinterstitial regions 172, a PAZAM layer 186 is deposited and/or formedon the coupling agent 176 (stage 190). As depicted, the PAZAM layer 186fills in the wells 170 and covers the interstitial regions 172. ThusPAZAM can conform to surface contours having appropriately sizedfeatures. For example, wells having an opening with a cross section thatis greater than about 100 nm² can be filled with PAZAM. It iscontemplated that wells having smaller cross sections can be used aswell under conditions where PAZAM fills the well or alternatively coversthe well without entering the space of the well. After application oflight (stage 200) to facilitate linking of the PAZAM layer 186, thephotoresist layer 162 is lifted off (stage 202), leaving only attachedPAZAM base pads 68. In the depicted embodiment, excess PAZAM in thewells 170 that is unlinked is also lifted off with the photoresist.

In certain embodiments, passivating the interstitials between the pads68 may prevent nonspecific binding during capturing, sequencing or otherapplications. That is, in addition to forming a desired pattern ofactive base pads 68, the interstitial spaces may be treated todiscourage undesired molecule binding. FIG. 23 illustrates an example inwhich lithography, or another patterning method, is employed to blocksections of the surface 90 of the substrate die 28 to create inert pads.As illustrated, after patterning a photoresist layer 220 via lithography(step 224), a passivation material 226 is applied at step 230 to theinterstitial spaces 228 exposed after patterning. Such passivationmaterials may include, but are not limited to, diamond-like carbon,polyethylene glycol, hexa-methyldisilizane, Teflon, and/or Parylene. Theapplication of the passivation material 226 and subsequent liftoff (step231) of the remaining photoresist later 220 yields a patterned surfacewith inert pads 232 forming the negative space of the desired pattern.The base pads 68 of any desired polymer may then be applied (step 236)to the surface 90 of the substrate die 28

In an alternative approach, the surface 220 of the substrate die 28 maybe passivated via metal patterning. In the approach illustrated in FIG.24, a metal patterning sublayer 240 protects the interstitial duringdeposition. The metal patterning sublayer may be formed from one or moreof aluminum, gold, titanium, as well as other metals. Metal can act as aphoto- and chemical mask during the surface linkage step, and theliftoff of the metal is a chemically simple procedure, which mayeliminate manufacturing steps relative to other processes. The metallayer 240 may include a photoresist layer 242 on an outermost surface.After APTMS and resist liftoff at step 248, the surface is ready forapplication of, for example, a PAZAM spin coat at step 254. The PAZAMlayer is cross-linked, for example via backside illumination through thedie 28, at step 260, and the base pads 68 (PAZAM pads in the depictedembodiment) are exposed after metal liftoff at step 264.

FIG. 25 is an example of selectively functionalized wells 270 used forapplying a polymer 272 only in the wells. For example, wells 270 may befunctionalized with covalent linkage methods or the polymer may benoncovalently lodged in the surface. The fabrication of wells 270 offersa simpler approach to surface functionalization. In one embodiment, apassivation layer may be applied only to the tops 274 of theinterstitial regions 276 to keep the top surface clean if necessary.Pregrafted or ungrafted PAZAM, or other polymer, may be applied.

While certain disclosed embodiments related to selectively patterning asurface with appropriate sites 22 (e.g., polymer pads 68), either withor without grafted primers, another approach may involve laying down asurface of polymers with associated primers and then selectivelyremoving, deactivating, decomposing or, otherwise rendering unusable theprimers from selected regions of the surface. Further, while thedisclosed techniques may be used alone to generate a patterned surface,they may also be used in conjunction with other disclosed patterningtechniques (e.g., base pad formation techniques) to yield a complexpatterned surface. In one embodiment, electrical fields may be used toselectively decompose nucleic acids at a particular region of a surface,repel nucleic acids from a particular region of a surface or removenucleic acids from a particular region of a surface to yield a desiredprimer pattern. The region of the surface from which nucleic acids aredecomposed, removed or repelled can be the interstitial regions betweenthe pads where nucleic acids are desired. For example, as illustrated inFIG. 26, an electrical current is applied to a lawn of primers 300. Inparticular, an electro-active surface 302 (such as, but not limited to,ITO) is decorated with dielectric pads 304 (such as, but not limited to,SiO₂) that act as resists. Grafted PAZAM, or other polymer 306, sitsatop this surface via covalent or non-covalent immobilization. Anelectrical current, or voltage potential, is applied through theelectrically conductive layer 302, resulting in the removal, ablation ordeactivation of the DNA primers present in those regions 310 withoutdielectric pads 304. Regions shielded by the dielectric pads 304 willretain features of PAZAM, or other polymer, with grafted primers 300.

FIG. 27 illustrates a liftoff approach for patterning primers. Aphotoresist layer 310, or other patternable substance, is deposited overa reactive layer 312 (e.g., PAZAM, or another polymer). The photoresistlayer 310 is then patterned via photolithography, nanoimprint or otherviable process at step 316. The primer grafting solution is flowed overthe top at step 320, resulting in restricted functionalization andapplication of primers 322, via homogenous or heterogeneous methods. Thepatterned photoresist layer 310 protects the interstitial regions 324 ofPAZAM, or other polymer, from reacting. Liftoff can then be performed atstep 330, leaving patterned areas of grafted primers 322.

A number of photoactivated/photocleaved grafting events may be performedto leave grafted primer lawns. In one example, illustrated in FIG. 28, asubstrate 28 that includes a photoactivatable covalent coating is seededat step 352 with photocleavable primers 354. In particular, aphotocleavage site 356 may be placed into the DNA and a photomask 357applied to yield a desired primer pattern at step 358. After irradiationat step 360, after irradiation, those regions not protected by thephotomask 357 are cleaved to yield cleaved non-reactive primers 362 andreactive primers 364. Alternatively, in another embodiment reactiveprimers may be protected by a photocleavable unit. Areas exposed tolight are released and made reactive leaving behind reactive primerregions and non-reactive primer regions.

In another embodiment, nanostructures may be used to faciliate base padformation. In one aspect, nanodots that are undersized relative tofabricated wells may be modified with a thick padding layer such thatthe whole structure is of a size that may be loaded singly into wellsfabricated by conventional lithography techniques. In one embodiment,nanodots are prefabricated (e.g. via sol-gel reduction, reduction from asalt solution, reduction from a micelle solution etc.) or purchased froma commercial vendor. Long polymers can be attached to thesenanoparticles using a specific interaction on one end of the polymer. Incertain embodiments, the polymer shell may be made very rigid bychemical crosslinking or by a solvent exchange leading to anentropically locked glassy state. As shown in FIG. 29, for goldnanoparticles, the specific interaction may be to a thiol group presenton one end of the polymer. In another embodiment in which titanium oxidenanoparticles are used, the specific interaction may be due to acarboxylic acid termination on one end of the polymer. To increase thesize of the polymeric shell, the polymer is added to the nanoparticlesin a suitable solvent. In solvent, the polymer is stretched, ensuringboth a dense and a thick shell around the nanoparticles. The shell maybe crosslinked for stiffness. Any free double bonds may be crosslinkedphoto-chemically using light activation of a photochemical crosslinkingreagent or moiety, or crosslinked chemically using any of a number ofsmall molecule crosslinking reagents or moieties. Alternately, thenanoparticle-shell solution may be diluted into a non-ideal or thetasolvent of the polymer, forcing the dense polymer shell to collapse,resulting in a glassy, sterically locked conformation. It is to beunderstood that the polymer shell could consist of a homopolymer ormutiple-block-copolymers, wherein further, the binding to thenanoparticles could be due to specific interactions with one of theinner blocks of the co-polymer micelle, as shown in FIG. 30. Forexample, the precursors to nanoparticles, preferrably metal salts ormetal alkoxides in a solvent medium can be chelated in the cores of acopolymer micelle. This combines the processes of nanopartice synthesisand deposition, allowing more control over both processes. Briefly, thecore of the micelle is a polymer that is able to complex with the metalor is able to sequester the metal solution due to surfactant actionprotecting it from the solvent. The disclosed techniques may be used tocrosslink or stiffen the micelles, if needed.

As shown in FIG. 31, nanoparticle-containing shells, such as thoseexemplified above, may be loaded in to large photoresist wells on asubstrate. Once loaded, the polymer is burned off in a plasma chamberand the photoresist removd in a suitable solvent leaving singlenanoparticles in an ordered array. The nanoparticle-precursor containingmicelle can be deposited into nanowell arrays by spin coating ordipcoating. Reduction of the metal solution to metal can occur underoxygen plasma or high temperatures, which destroys the polymeric micellein the process as well. The nanowell arrays are typically produced bystandard lithography techniques, wherein one embodiment is nanowellsproduced in photoresist layers which may be stripped away after thenanoparticle reduction. In addition, passivation techniques may be usedin conjunction with the above nanostructure embodiments, or any otherdisclosed embodiments. In particular, entaglement of library elements tothe SFA matrix and the non-specific binding of DNA capture moities(avidin) on the flowcell surface may conrtibute to non-specificbackground noise. In one embodiment, a diamond-like carbon (DLC)passivation layer is applied to all or part of a flowcell surface. DLCcan be easily etched and processed with standard lithography tools.Further, DLC is hydrophobic and biocompatible. The passivation layer caninclude other materials including. For example, hexa-methyldisilizane,Teflon, fluorocarbons, parylene, perfluorinated polymers, metals, metaloxides, or PEG or other types of passivating polymers.

In another embodiment, a DLC film or mask may be used to grow DNAclusters in predetermined positions as well as control the size of theclusters by confining their growth to the size of the patterned feature.The pattern of DLC impedes both DNA templates seeding and amplification.In one example, a 30 nm thick DLC film was deposited on glass flowcellsubstrates and windows on the DLC film were opened only at desiredpositions. Using the DNA seed-through biochemistry process, DNAtemplates were only seeded in the windows in the DLC film, and the DNAclusters were confined within the window area after bridge amplificationprocess. FIG. 32A shows a fluorescent image of SYBR Green-stainedcluster formed on a DLC-patterned substrate, and FIG. 32B shows a 1^(st)base image of the cluster array of FIG. 32B.

This DLC based cluster growth control system faciliates patterning ofhighly ordered cluster arrays that increase area cluster density andsimplify signal analysis processes to boost the sequencing throughput.The DLC can also be applied to existing flowcell products more generallyto deplete the unwanted cluster growth; for example, on the top channelsurfaces for one-side imaging system. Besides the glass, the DLC canalso be patterned on different dielectric substrates such as Si₃N₄ orSiO₂ coated Si substrates.

In one example, illustrated in FIG. 33, a DLC film is made at step 300.The film can be made by plasma enhanced chemical vapor deposition(PECVD) onto glass, which may include systems with methane (CH₄) as thegas source. After application of a photoresist layer at step 32, etchingat step 304, and liftoff at step 306, a patterned DLC layer 310 may beformed. In addition, the surface energy of the DLC film can be tailoredby adding CF compound gas during PECVD. Chemical surface modification ofDLC by using 3-Aminopropyltriethoxysilane (APTES) can also be used inDLC patterned flowcells.

In addition to patterning techniques, improved binding performance forany type of sequencing or other biological reaction, such as thosedisclosed herein, may be achieved by altering the characteristics of thereaction solution or the reaction conditions to encourage molecularcrowding, which may result in enhanced binding at the sites 22. In oneembodiment, the disclosed substrates and arrays may be used inconjunction with molecular crowding techniques. Briefly, when twomacromolecules are mixed in a solution, the free energy of mixingpromotes the miscibility of the two populations whereas thetranslational entropy is maximized when the two components are phaseseparated. If one of the components of the mixture is capable ofrestricting the free motion of the second component, the depletioninteraction is pronounced, leading to domains of like-molecules withgreatly increased local concentrations. Adding suitable concentration ofPEG solutions of an appropriate molecular weight may concentratetemplate molecules within the flowcell leading to an enhanced rate ofcapture at the sites 22. FIG. 34A-B show results from an experiment inwhich PEG was used to improve seeding efficiency for avidin andbiotin-labeled DNA interaction. In both the control and the PEG crowdedrun, 0.015 mg/ml of avidin was non-specifically bound to the surface ofan unpatterned flow cell. Biotin-labeled (i.e., P5 end labeled) DNA wascontacted in the absence of (FIG. 35A) and presence of (FIG. 35B) 5% PEG8000 solution. Images were taken of clusters from a G channelacquisition on a HiSeq 2000 (Illumina, Inc., San Diego Calif.). WithoutPEG, as shown in FIG. 34A, the run achieved 98.3% alignment with 95.6%rate pass filter and about 200K/mm2. With PEG, as shown in FIG. 34B, therun achieved 95.2% alignment with 83.4% rate pass filter and greaterthan 900 K/mm². The reaction with PEG exhibited greater cluster density.Accordingly, it is contemplated that the present techniques mayincorporate PEG or other reaction solutions that faciliate molecularcrowding. In one embodiment, the substrates and/or microarrays discloedherein may be used with about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,25% or 50% PEG. Further, PEG may be present in the reaction solution orthe flowcell solution in ranges of about 1%-10%, 1%-8%, 1%-3%, 3%-5%, or5%-10%, 1%-25%, 1%-50% or 10%-50%.

In another embodiment, electrophoresis may be used to concentrate DNAmolecues close to the surface. In one implementation, a transparentconductive layer such as indium tin oxide (ITO) is coated on the top andbottom surfaces of the flowcell such that the ITO surfaces function aselectrodes. An applied electric field drives the DNA molecules towardsthe surfaces/electrodes, where they are specifically immobilized to thecapture pads. Over time the DNA molecules adhere to the surfacesnon-specifically, whereas in the absence of the field, no such surfaceaccumlation and adsorption is seen. In addition to ITO surfaces, othertype of electrically-conductive surfaces may be appropriate forencouraging molecular movement towards the substrate 28, such as oxideor polymer surfaces. Exemplary surface materials iclude, but are notlimited to, SnO₂, aluminium-dope ZnO (AZO), ZnO, TiO₂, Poly(3,4-ethylenedioxythiophene (PEDOT), and the like. In one embodimentthat uses a oscillating electric field, the DNA molecules concentrate tothe top and bottom surfaces cyclically. The oscillating field providesan additional benefit of reducing electrolysis and mimizingelectrochemisty at the surfaces.

FIG. 35 illustrates a workflow for creating gold pads on an ITO surface.After evaporation of ITO to form an ITO layer 320 (e.g., 200 nm thick)on the surface of the substrate 28, a double-layer resist 322 isspin-coated into the ITO layer 320 and etched at step 324. Thedouble-layer photoresist layer 322 may be used to achieve clean edges ofthe pads. Ti/Au deposition, for example via evaporation, is performed atstep 326 to yield an Au layer 330 in the interstitials 332 of thephotoresist layer 322. In one embodiment, the Au layer 330 may be about60 nm or less while the Ti layer may be evaporated to about 4 nm orless. Following a liftoff at step 340, the substrate die 28 can bepatterned according to a desired pad size and pitch. At step 350, theAu-patterned substrate die 28 and a plain ITO surface 360 are sandwichedinto a flowcell 362, which may include appropriate casing and spacercomponents, such as outer layer 370 and spacers 380. A 4 voltpeak-to-peak (+2 to −2) voltage at 0.5 Hz can be used to draw themolecules towards the top and bottom surfaces cylically. FIG. 36 is acircuit diagram of an exemplary circuit that may be used to provide ACsignal to the flowcell 362. FIG. 37 is an exploded view of the flowcell362. As shown, the substrate 28 and the outer layer 370, which, incertain embodiment, may be glass and/or the same material as thesubstrate layer 28, have respective notches 398 and 400. As illustrated,the notches are at opposing corners. However, it should be understoodthat the notches may be positioned in any suitable location to permitaccess to the ITO layer 360 and the ITO layer 320 so that AC power maybe supplied across the flow cell 362. Similarly, the spacer 380 also mayinclude notches 402 and 404 that are aligned with notches 398 and 400,respectively.

In addition to transverse electrical pulldown, a longitudinaltime-varying electric filed across interdigitated electrodes may also beused to concentrate DNA by dielectrophoresis. Dielectropheresis issensitive to mass. Therefore, a size-dependent pulldown of DNA can beachieved by manipulating the dielectrophoretic force. Thedielectrophoretic force increases by decreasing the spacing between theinterdigitated electrodes and also by increasing the applied field andfrequency of oscillation. Large molecules experience larger forces atlow field and frequency, while smaller molecules are pulled down bylarger and high-frequency oscillating fields. A DEP based pulldown canremove the need for size selection of libraries while also allowingapplications such as pulling down protein-bound DNA to the surfaceselectively (for example, to accommodate real-time field-sortedchromatin immunoprecipitation sequencing (CHIPSeq)). FIG. 38 is anexample of a flow cell configuration that may be used for DEP. Theflowcell 418 includes passive areas 420 separating a serpentineelectrode 422 that are applied to the substrate 28. The electrode ispowered via a voltage source 430. FIG. 39 is a schematic illustration ofthe generation of dielectric forces between the areas of the electrode422.

As discussed herein, the microarrays disclosed herein facilitate bindingand/or amplification of a single molecule (e.g., steric exclusion orkinetic exclusion such that only one molecule is copied at each pad orfeature of an array). Typically the patterns contain a DNA capturemoiety and the DNA molecules contain a binding moiety (e.g.,streptavidin incorporated into base pads and biotin on the DNA). If thenumber of binding moieties on the DNA is equal or greater than thenumber of capture moieties on the pad, one, and only one, DNA moleculecan bind to a pad. This is in addition to steric repulsion, which canitself help in reducing multiple bindings to the same pad. In certainembodiments, capture of template DNA molecules is a two stage process.For example, avidin molecules are first immobilized onto gold pads viathiol bonds and DNA containing biotin on one end are captured by theavidin on the gold pads. There are four biotin binding sites per avidin,and there are multiple avidins per gold pad. Steric hindrance mayprevent multiple DNA molecules from binding to the same gold pad. Sterichindrance is improved if the sites (e.g., base pads 68) are very small.However, there is a possibility of inducing multiple bindings per pad.One technique to ensure clonality of seeding is to ensure the first DNAmolecule that binds to a pad is able to saturate all theDNA-capture-moieties on the pad.

In one embodiment, multidentate ligands or receptors may be used toincrease the number of binding moities on DNA that binds to a pad.Exemplary multidentate ligands or receptors that can be used include,but are not limited to, dendrons, avidin, streptavidin and functionallyactive derivatives thereof. In one embodiment, a dendron (or othermultidentate ligand or receptor) is incorporated into the librarythrough a PCR primer or through a transposome complex in the case of PCRfree libraries such as those used in TruSeq Nextera protocols availablefrom Illumina Inc. (San Diego, Calif.). Either P5, P7 or both P5 and P7can be modified with a dendron (or other multidentate ligand orreceptor) on their 5′ end (e.g. 5′ azide followed by click reaction withacetylene on the Dendron). Multidendate ligands or receptors with —COOHmoieties can directly bind to TiO₂, ZnO, Al₂O₃, and ITO nanodots. Thecarboxyl group can be converted to a biotin or thiol using abifunctional PEG linker. Further, the reach of the arms of the ligandsor receptors can be increased by adding PEG spacers, allowing a singletemplate molecule to access/bind-to a large surface area of the capturepad via the multiple receptors or ligands. A thiol terminated dendron(or other multidentate ligand or receptor) can be used to directly bindto the gold pads without needing the intermediate avidin layer. As shownin FIG. 40, a commercially available dendron is attached to a primer andis then converted to include a desired end group (biotin, thiol, etc.).An advantage of using multidentate ligands is increased stability(exponential with addition of binding groups) and increased kinetics ofseeding of DNA on pads compared to use of single ligands.

Particular embodiments, involve using mulitidentate ligands or receptorsengineered into avidin and DNA. These constructs can be used to seed DNAto a pad directly or via a sandwich avidin/biotin DNA construct. Thesemethods take advantage of the increased avidity and binding stability inmetal-ligand interactions (ZnO, ZnS, Gold) with multidentate ligands.Alternatively or additionally to carboxylic acid moieties inmultidentate ligands set forth above, multiple thiols, phosphines,phosphine oxides, or amines (NH₂) can be used to bind nucleic acids topads. Such moieties can be incorporated into nucleic acids, for example,by using chemically modified primers to produce modified amplicons in aPCR reaction or by chemical modification of nucleic acids using knownchemistrires such as N-hydroxy succinimide (NHS) reactions. In additionto dendrons, multi arm PEGs (e.g having greater than 2 arms) can be usedto covalently link binding groups to nucleic acids. Proteins such asavidin or streptavidin can be attached to nucelic acids via NHSreactions reactions.

Multidentate lignads and receptors can be used in combination withelectric field assisted seeding of nucleic acids to pads. For example,multidentate ligands or receptors may be used to increase the number ofbinding moities on nucleic acids that binds to a pad and an appliedelectric field can be used to drive the nucleic acid molecules towardsthe surfaces/electrodes, where they are specifically immobilized to thecapture pads via the multidentate ligands or receptors.

In an alternative embodiment, amine-labeled nucleotides in the primercan be functionalized with an NHS-PEG. Adding binding moities to bothends of the template molecules is another way to improve clonality (i.e.homogeneity of amplicons at an individual pad or feature of an array).As shown in FIG. 41, capturing from both ends of the template may reducethe number of cycles needed to form clusters of a given size. As shown,the primer lawn includes primers that terminate with a characteristicsequence at one end and a different characteristic sequence at the otherend. The difference characteristic sequences may include those availablefrom Illumina, such as the P5 adapter and the P7 adapter, which form aprimer lawn 450. In one embodiment, the primer terminates with P5 at oneend and P7 at the other. After cluster seed formation, self-repellingclusters form because the cluster seeds are complementary strands. Ineach cluster, the complementary strand of the cluster seed has a P5anchor, including a U, shown as region 452. Specific cleavage of the P5results in clonality. The sequences for P5 and P7 adapters are set forthin Bentley et al., Nature 456:53-59 (2008) and WO 00/31148, each ofwhich is incorporated herein by reference in its entirety.

Even if the DNA and/or avidin bind non-specifically, if any clustersthat form only grow around the sites 22, the issue of non-specificclusters can be avoided. In the embodiment shown in FIG. 42, the endsequences of the template are P5 and P6. These templates cannot clusteron the SFA lawn which contains immobilized primers P5 and P7. A ‘P6-P7’primer is immobilized on sites 22. This primer allowshybridization-extension-copy of the captured template by providing acomplement to the 5′ end of the molecule which is not present elsewhereon the SFA matrix. This primer also provides the P7 anchor needed forcontinued copy and clustering cycles that can proceed on the SFA matrixaround the sites 22 (e.g., a nanodot site). The P6 sequence may be anSBS sequencing primer (e.g., SBS3). This method provides a robust andsimple process to avoid non-patterned clusters of any kind. For the caseof SFA-entangled DNA that is not necessarily bound to any avidin or abinding moiety, a 5′ exonuclease such as lambda exonuclease may be usedto chew back from the 5′ end of the DNA. For molecules bound to thecapture pad or biotin, the 5′ end will not be accessible to the nucleasebecause the binding/capture occurs from the 5′ end of the molecule. Asshown in FIG. 43, DNA at the sites 22 is able to form a cluster whileDNA in the interstitial spaces does not form any clusters. The sequencesfor the primers and other oligonucleotides identified above are setforth in Bentley et al., Nature 456:53-59 (2008) and WO 00/31148, eachof which is incorporated herein by reference in its entirety.

FIG. 44 illustrates system components generally in an overall system 80for making, preparing and utilizing microarrays of the type described,along with certain operations performed by the system components. Thesystem may be considered to include an array preparation system 482, anarray reading system 484, and an analysis system 486. These threesystems may be present as components of a larger system as exemplifiedin FIG. 44. Alternatively one or more of systems 482, 484 or 486, orcomponents thereof may be present in separate systems. Furthermore,various components exemplified in FIG. 44 may be optionally omitted insome embodiments. The preparation system may begin with a microarray ofthe type described above, adapted for capture of a molecule at eachsite. Moreover, as mentioned above, the microarray will typically bedisposed in a flow cell, and in certain embodiments, more than onesurface within the flow cell may be configured to receive molecules ofinterest at the sites provided.

As indicated at step 488, then, the exemplary system may be operated toallow for molecule capture. This process can involve flowing a desiredconcentration of the target molecules through the flow cell in which thearray is positioned. In certain presently contemplated implementations,for example, segments of DNA or RNA may include primers at either end,with an attachment molecule, such as biotin secured to at least one ofthe primers. Owing at least in part to the small size of the sites, andpossibly to other effects, such as steric and charge hindrance, eachsite will preferably only attract and/or attach a single molecule.However, in other embodiments, the sites may be generally larger and maybe capable of capturing a plurality of molecules. As noted above, thecapture substance provided at each site serves to hold the molecule ofinterest. The molecules are then amplified, as indicated at step 490.While several different amplification techniques may be utilized, in apresently contemplated implementation, bridge amplification isparticularly useful. This and other amplification techniques may becarried out using techniques known in the art as described in referencesset forth previously herein. Amplification allows for a large number ofidentical molecules to be co-located at each site, thereby significantlyimproving the robustness of the subsequent processing, and enhancingsignal-to-noise ratios. The flow cell may then be prepared for imagingand analysis, as indicated by reference numeral 492. This process willtypically involve connecting the flow cell to inlet and outlet conduitsfor the flow of nucleotides or other chemistry, as well as for the flowof deblocking agents, flushing agents, and so forth. The flow cell mayalso be positioned in a processing/imaging arrangement that forms partof the reading system 484. Such may provide for fully or semi-automated,and where desired, cyclic processing and imaging of the sample. Suchsystems are described in U.S. Pat. No. 7,329,860; U.S. patentapplication publication nos. US 2010/0111768 A1, or 2011/0220775 A1; orU.S. Ser. No. 13/273,666 or 13/006,206, each of which is herebyincorporated by reference in its entirety.

The reading system 484 may employ a bio-molecule reagent delivery systemfor delivering various reagents to a sample as it progresses through thesystem, as indicated by reference numeral 494. The particularconfiguration of such systems, their degree of automation, the number ofcycles the sample may be imaged, and the particular chemistry involvedwill, of course, depend upon the nature of the molecules beingevaluated, as well as the system design. In general, system may includea plurality of stations through which samples and sample containers(e.g., flow cells) progress. This progression may be achieved in anumber of ways including, for example, physical movement of the sampleto different stations, physical movement of different stations to asample, delivery of fluid from different stations to a sample such asvia valve actuation or some combination thereof. A system may bedesigned for cyclic operation in which reactions are promoted withsingle nucleotides or with oligonucleotides, followed by flushing,imaging and de-blocking in preparation for a subsequent cycle, asindicated by reference numerals 496, 498 and 500. In a particularsystem, the samples may be circulated through a closed loop path forsequencing, synthesis, ligation, or any other suitable process. Again,it should be noted that the process illustrated is not necessarilylimiting, and the present invention may allow data to be acquired fromany suitable system employed for any application (e.g. image data,electrical data etc.).

In the illustrated embodiment, the nucleotide delivery operation 494provides a process stream to the samples. An effluent stream from theflow cells may be discarded or, if desired, recaptured and recirculatedin the nucleotide delivery system. In the illustrated embodiment, then,the sample container may be flushed in the flush operation 496 to removeadditional reagents and to clarify the sample for imaging. The sample isthen imaged or otherwise detected in the data capture operation 490where data may be generated that may be analyzed for determination ofthe sequence of a progressively building nucleotide chain, such as basedupon a template, or for any other analysis, depending again upon thenature of the molecules. In a presently contemplated embodiment, forexample, an imaging system used for this operation may employ confocalline scanning to produce progressive pixilated image data that may beanalyzed to locate individual sites in an array and to determine thetype of nucleotide that was most recently attached or bound to eachsite. Other imaging techniques may also suitably be employed, such astechniques in which one or more points of radiation are scanned alongthe sample. Various embodiments of the systems and methods of thepresent disclosure are exemplified with respect to optical detection. Itwill be understood that other detection modes (e.g. non-opticaldetection) may be used. For example, sequencing based on detection ofreleased protons can use an electrical detector and associatedtechniques that are commercially available from Ion Torrent (Guilford,Conn., a Life Technologies subsidiary) or sequencing methods and systemsdescribed in US 2009/0026082 A1; US 2009/0127589 A1; US 2010/0137143 A1;or US 2010/0282617 A1, each of which is incorporated herein by referencein their entireties. Some embodiments can utilize nanopore sequencing,whereby target nucleic acid strands, or nucleotides exonucleolyticallyremoved from target nucleic acids, pass through a nanopore. As thetarget nucleic acids or nucleotides pass through the nanopore, each typeof base can be identified, for example, by measuring fluctuations in theelectrical conductance of the pore (U.S. Pat. No. 7,001,792; Soni &Meller, Clin. Chem. 53, 1996-2001 (2007); Healy, Nanomed. 2, 459-481(2007); and Cockroft, et al. J. Am. Chem. Soc. 130, 818-820 (2008), thedisclosures of which are incorporated herein by reference in theirentireties).

Following the detection and data collection operation, then, the samplesmay progress to a de-blocking operation 500 in which a blocking moleculeor protecting group is cleaved from the last added nucleotide, alongwith a marking dye. If the system is used for optically detectedsequencing, by way of example, image data may be stored and forwarded toa data analysis system as indicated generally at reference numeral 484.

The analysis system will typically include a general purpose orapplication-specific programmed computer providing for user interfaceand automated or semi-automated analysis of the data to determine whichof the four common DNA nucleotides was detected as a particularsequencing cycle (e.g. in the case of SBS, the identifying of thenucleotide that was last added at each of the sites of the array can bedetermined). As will be appreciated by those skilled in the art, in someembodiments such analysis may be performed based upon the color ofunique tagging dyes for each of the four common DNA nucleotides. Thedata may be further analyzed by the downstream data analysis operations502 and processing and data storage operations 504. In these operations,secondary data derived from the primary data may be stored, encoded,processed and analyzed. Due to the large volume of data collected,certain portions of the primary or secondary data may be compressed ordiscarded. Again, the sequencing application is intended to be oneexample only, and other operations, such as diagnostic applications,clinical applications, gene expression experiments, and so forth may becarried out that will generate similar data operated on by the presentinvention. Some examples of array based methods that generate image datathat may be made and used in accordance with the teachings hereininclude, array-based genotyping or expression analyses. Such analysesmay be carried out, for example, based on binding of a labeled targetanalyte to a particular probe of the microarray or due to atarget-dependent modification of a particular probe to incorporate,remove, or alter a label at the probe location. Any one of severalassays may be used to identify or characterize targets using amicroarray as described, for example, in U.S. Patent ApplicationPublication Nos. 2003/0108867 A1; 2003/0108900 A1; 2003/0170684 A1;2003/0207295 A1; or 2005/0181394 A1, each of which is herebyincorporated by reference in its entirety. It is contemplated that thesystem, or various subcombinations of the exemplified system components,may include an interface designed to permit networking of the system toone or more detection systems acquiring image data (or other data) frombiological microarrays of the type described. The interface may receiveand condition data, where appropriate. In general, however, an imagingsystem will output digital image data representative of individualpicture elements or pixels that, together, form an image of thebiological microarray. One or more processors process the received imagedata in accordance with a plurality of routines defined by processingcode. The processing code may be stored in various types of memorycircuitry, and will include informatics routines for determining thenature of the molecules captured at each site of the array, and wheredesired, for determining possible structures comprising these (e.g.,piecing the molecules together in longer, meaningful groups.

While only certain features of the contemplated embodiments have beenillustrated and described herein, many modifications and changes willoccur to those skilled in the art. It is, therefore, to be understoodthat the appended claims are intended to cover all such modificationsand changes as fall within the true spirit of the disclosure.

1. A method for preparing a biological microarray, comprising: providingan array of base pads at predetermined sites on a substrate, whereinindividual base pads are configured to capture a nucleic acid molecule;and contacting the array of base pads with a mixture of differentnucleic acid molecules under conditions wherein a nucleic acid moleculeis captured at each base pad, wherein a porous attachment layer isdisposed over the base pads and the porous attachment layer isconfigured to attach amplified copies of the nucleic acid moleculescomprising nucleotides or nucleotide-like components.
 2. The method ofclaim 1, wherein individual base pads are configured to capture no morethan one nucleic acid molecule.
 3. The method of claim 2, wherein asingle nucleic acid molecule is captured by each and every base pad inthe array.
 4. The method of claim 2, wherein a single nucleic acidmolecule is captured by fewer than all of the base pads in the array. 5.The method of claim 1, wherein the nucleic acid molecules comprisenucleotides or nucleotide-like components.
 6. The method of claim 1,further comprising amplifying the single nucleic acid molecule at eachbase pad to obtain at each base pad a region comprising copies of thenucleic acid molecule, wherein the copies of the nucleic acid moleculeare attached to the porous attachment layer.
 7. The method of claim 1,wherein the substrate comprises a glass.
 8. The method of claim 1,wherein the base pads comprise gold.
 9. The method of claim 1, whereinthe base pads are formed by an imprint lithographic process.
 10. Themethod of claim 9, wherein the imprint lithographic process comprisesdisposing a transfer layer on the substrate, disposing a light-curingresist layer over the transfer layer, patterning the resist layer via amold, curing the patterned resist layer, etching the resist and transferlayers to expose regions of the substrate, depositing a pad-formingsubstance over the exposed regions of the substrate, and removing theresist and transfer layers.
 11. The method of claim 10, wherein curingthe patterned resist layer comprises exposing the mold to radiation thattraverses the mold to cure the patterned resist layer without removingthe mold from the patterned resist layer.
 12. The method of claim 1,wherein the porous attachment layer is patterned.
 13. The method ofclaim 12, wherein the porous attachment layer comprises a plurality ofregions at least partially surrounding each of the base pads.
 14. Themethod of claim 1, wherein the porous attachment layer extendssubstantially continuously over the plurality of base pads.
 15. Themethod of claim 1, wherein the porous attachment layer comprisesacrylamide.
 16. A method for preparing a biological microarray,comprising: forming an array of base pads at predetermined sites on asubstrate; disposing a molecule binding substance over each of the basepads, thereby configuring each of the base pads to capture a nucleicacid molecule; disposing a porous attachment layer over the base pads;seeding each of the base pads with a single nucleic acid molecule bylinking the single nucleic acid molecule to the molecule bindingsubstance; and amplifying the nucleic acid molecule at each base pad toobtain at each base pad a region comprising copies of the nucleic acidmolecule, wherein the copies of the nucleic acid molecule are attachedto the porous attachment layer.
 17. A biological microarray system,comprising: an array of base pads at predetermined sites on a substrate;a molecule binding substance disposed over each of the base pads andlinked to no more than a nucleic acid molecule; a porous attachmentlayer disposed over the base pads; and several copies of each of thenucleic acid molecules linked to the porous attachment layer disposedover each of the respective base pads.
 18. A method for preparing abiological microarray, comprising: forming a polymer layer on asubstrate; disposing a photoresist layer over the polymer layer; forminginterstitial spaces in the photoresist layer and the polymer layer;removing the photoresist layer to expose polymer base pads, wherein thepolymer base pads are coupled to a molecule binding substance.
 19. Themethod of claim 18, wherein the polymer layer comprises apoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) polymer.
 20. Amethod for preparing a biological microarray, comprising: activatingregions on a substrate to form a pattern of activated regions;contacting the substrate with a self-assembling monomer solution;polymerizing the monomers to form polymer base pads only on theactivated regions wherein the polymer base pads are coupled to amolecule binding substance.
 21. A method for preparing a biologicalmicroarray, comprising: forming an electrically conductive layer on asurface of a substrate; forming a plurality of spaced apart electricallynonconductive regions on the electrically conductive layer; forming apolymer layer over the electrically conductive layer and the pluralityof spaced apart electrically nonconductive regions, wherein the polymeris coupled to a plurality of primers; and applying a current through theelectrically conductive layer to deactivate only a portion of theprimers coupled to the polymer.
 22. A method for preparing a biologicalmicroarray, comprising: forming a polymer layer on a surface of asubstrate; forming a plurality of spaced apart photoresist regions onthe polymer; contacting exposed portions of the polymer layer with aplurality of primers; and removing the photoresist regions and coveredportions of the polymer layer such that a plurality of spaced apartpolymer pads coupled the plurality of primers remain on the surface ofthe substrate.
 23. A method for preparing a biological microarray,comprising: forming wells on a substrate, wherein the wells areseparated by photoresist interstitial regions; applying nanoparticles onthe substrate such that the nanoparticles cover the wells and thephotoresist interstitial regions; removing the photoresist such that aplurality of spaced apart nanoparticles remain on the surface of thesubstrate; and coupling a molecule binding substance to thenanoparticles.
 24. A method for amplifying nucleic acids, comprising (a)providing an amplification reagent comprising (i) an array ofamplification sites, and (ii) a solution comprising a plurality ofdifferent target nucleic acids, wherein the different target nucleicacids have fluidic access to the plurality of amplification sites andwherein the solution comprises at least 3% PEG, and (b) reacting theamplification reagent to produce a plurality of amplification sites thateach comprise a clonal population of amplicons from an individual targetnucleic acid from the solution, wherein the reacting comprises (i)producing a first amplicon from an individual target nucleic acid thattransports to each of the amplification sites, and (ii) producingsubsequent amplicons from the individual target nucleic acid thattransports to each of the amplification sites or from the firstamplicon.
 25. A method for amplifying nucleic acids, comprising (a)providing an amplification reagent in a flow cell comprising (i) anarray of amplification sites, and (ii) a solution comprising a pluralityof different target nucleic acids, wherein the different target nucleicacids have fluidic access to the plurality of amplification sites, (b)appling an electric field across the flow cell to crowd the targetnucleic acids towards the array of amplification sites; and (c) reactingthe amplification reagent to produce a plurality of amplification sitesthat each comprise a clonal population of amplicons from an individualtarget nucleic acid from the solution, wherein the reacting comprises(i) producing a first amplicon from an individual target nucleic acidthat transports to each of the amplification sites, and (ii) producingsubsequent amplicons from the individual target nucleic acid thattransports to each of the amplification sites or from the firstamplicon.